Guenter Weigel

Tissue engineering of heart valves : Decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells

Background—Tissue-engineered or decellularized heart valves have already been implanted in humans or are currently approaching the clinical setting. The aim of this study was to examine the migratory response of human monocytic cells toward decellularized porcine and human heart valves, a pivotal step in the early immunologic reaction. Methods and Results—Porcine and human pulmonary valve conduits were decellularized, and migration of U-937 monocytic cells toward extracted heart valve proteins was examined in a transmigration chamber in vitro. Homogenized tissue specimens were size fractionated by SDS-PAGE. The decellularization procedure effectively reduced the migration of human monocytes toward all heart valve tissue. However, only the antigen reduction of human pulmonary valves abolished the monocytic response (wall, 0.88±0.19% versus 30.20±3.93% migrated cells [mean±SEM]; cusps, 0.10±0.06% versus 10.24±1.83%) and was significantly lower (P<0.05) than that of the decellularized porcine equivalent (wall, 5.03±0.14% versus 24.31±2.38%; cusps, 3.18±0.38% versus 10.24±1.83%). SDS-PAGE of the pulmonary heart valve tissue revealed that considerable amounts of proteins with different molecular weights that were not detected in the human equivalent remain in the decellularized porcine heart valve. Conclusions—We describe for the first time that the remaining potential of decellularized pulmonary heart valves to attract monocytic cells depends strongly on whether porcine or human scaffolds were used. These findings will have an important impact on further investigations in the field of heart valve tissue engineering.

Electrospun polyurethane vascular grafts: In vitro mechanical behavior and endothelial adhesion molecule expression

Engineered small diameter vascular grafts must closely match mechanical characteristics of native vessels and exhibit stimulus-responsive bioactivity. In this study, mechanical homogeneity of electrospun small diameter polyurethane grafts as well as spontaneous attachment, proliferation, and adhesion molecule expression of endothelial cells (EC) in their presence was studied in vitro. Axial and circumferential tensile strengths were measured and found to be twofold higher in the circumferential direction. EC attachment was easily achieved without precoating the fiber matrix. Stimulation of EC with interleukin-1beta (IL-1beta) led to a statistically significant upregulation of the adhesion molecules E-Selectin, ICAM-1, and VCAM-1. Quantification of adhesion molecule expression by means of energy-dispersive X-ray microanalysis revealed no differences in the stimulatory responses of EC cultured on electrospun polyurethane when compared with cells grown on tissue culture-treated cover slips. Summarizing, highly uniform small diameter polyurethane grafts were fabricated and shown to allow spontaneous EC attachment. The synthetic graft surface neither impaired the endothelial response toward IL-1beta stimulation nor did it adversely affect the regulation of expression of endothelial adhesion molecules.

Electrospun Small‐Diameter Polyurethane Vascular Grafts: Ingrowth and Differentiation of Vascular‐Specific Host Cells

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.

Decellularized, xenogeneic small-diameter arteries: Transition from a muscular to an elastic phenotype in vivo

Reports regarding the biocompatibility of xenogeneic, decellularized bioprosthetic implants differ between bioinertness and complete graft degradation. We investigated heparin-crosslinked and nonheparinized, xenogeneic vascular substitutes in a rat model. Porcine arteries (15 x 1.5 mm) were decellularized by multistep detergent and enzymatic techniques, which were followed by heparin-crosslinking in 50% of the implants. Prostheses were implanted into the abdominal aorta of 76 rats for 1 day and up to 6 months. Retrieved specimens were evaluated by histology, immunohistochemistry, laser scanning, and scanning electron microscopy. Graft patency did not differ between groups (97.3%). Heparinized grafts showed a statistically significant lower rate of aneurysm formation (p = 0.04 %). Implants revealed infiltration with granulocytes and macrophages up to 3 months. Recellularization with endothelial cells and myofibroblasts was detectable within 1 month. After 6 months elastin biosynthesis and complete graft remodeling toward an elastic vessel was evident. These results indicate that temporary inflammation does not interfere with long-term vascular remodeling.

(Bio)degradable Urethane-Elastomers for Electrospun Vascular Grafts

Electrospinning is a very powerful method to create cellular scaffolds for regenerative medicine – especially for artificial vascular grafts. Commercially available thermoplastic polyurethane elastomers (TPUs), like PellethaneTM are FDA approved and have already shown excellent biomechanical properties as electrospun vascular grafts. In order to induce the growth of a neo-artery and hence increase the long-term patency of the graft, the use of biodegradable TPUs is beneficial. Therefore we aim for the development of degradable TPUs. In preliminary studies the mechanical properties of segmented TPUs were examined. The tendencies of the properties of the compression-molded bulk materials were also found for the electrospun materials. It could also be shown that the substitution of the aromatic 4,4-methylene diphenyl diisocyanate building blocks in PellethaneTM with the aliphatic hexamethylene diisocyanate – to avoid toxic aromatic amines as degradation products only causes minor loss of strength. To obtain degradable TPUs, our concept is to incorporate cleavable ester bonds into the polymer chain. For this purpose, lacticand terephthalic ester-based cleavable chain extenders were used. The expected degradation products showed no cytotoxicity in-vitro. Degradation tests of polymer samples in phosphate buffered saline at elevated temperatures confirmed the degradability of the new polymers. INTRODUCTION Diseases of the cardiovascular system are one of the main causes of morbidity and mortality in the western hemisphere. Surgical therapy of cardiovascular disorders frequently requires the replacement of the diseased tissue with prosthetic grafts. Autologous vessels are the preferred replacement grafts, but many patients have no suitable vessels for harvest due to coexisting diseases or reoperation. The search for vascular substitute materials was thus directed at bioinert materials that minimally interact with blood and tissue. Elastic polymers, like expanded polytetrafluorethylene (ePTFE) or polyethylene terephthalate (PET) are currently the standard prosthetic materials which are used in vascular surgery. However, these synthetic materials have proved to be inferior to autologous conduits, especially when used for small caliber vessels or in low-flow applications. The main reasons for the poor performance are anastomotic intimal hyperplasia and innate surface thrombogeneicity. . In-vivo studies with grafts made of biodegradable thermoplastic polyurethane elastomers (TPUs) revealed that these materials promote the growth of neo-arteries and the graft therefore exhibits long-term patency [2]. But beside the chemical properties, the microstructure of the material plays an important role for the biocompatibility of the artificial grafts. Electrospinning (ES) has proofed to be the ideal processing tool for synthetic grafts as the random orientation of the nano-fibers perfectly mimics the extracellular matrix [3-5]. The three-dimensional scaffold serves as an framework for cell adhesion, proliferation and differentiation. Therefore we were interested in the development of new biodegradable TPUs. Generally, segmented TPUs possess two different moieties – the hardand the soft-blocks. Degradability can be introduced in each or both parts of the polymer through the incorporation of (hydrolytically or enzymatically) cleavable bonds (Figure 1). hard-block soft-block diisocyanate chain extender prepolymer association soft-block degradation cleavable bond hard-block degradation Basic composition of TPUs Introduction of degradability Figure 1. Concepts for degradable thermoplastic urethane elastomers. This study aims on the systematically, stepwisely conversion of the commercial TPU PellethaneTM (Dow) into a biocompatible, biodegradable elastomer that can be spun with ES to obtain artificial (cardio)vascular grafts (Figure 2). Therefore the different components (diisocyanate, prepolymer and chain extender) were substituted in order to improve the biocompatibility and induce degradability (Table I). reference material substitution of aromatic diisocyanate • mechanical study • electrospinning concepts for hard/soft-block degradability cytotoxicity tests of the expected degradation products synthesis of cleavable chain extender (CCE) and TPUs tests of new TPUs • degradability tests • mechanical tests optimization of ES process • mechanical tests • in-vitro cell tests Figure 2. Concept for the development of degradable polyurethane-based vascular grafts. Table I. Substitution of the components for the TPU synthesis. reference modification aim diisocyante CH2 OCN 2 MDI NCO OCN HMDI avoid aromatic amines as degradation products/metabolites chain extender O H OH BDO O

3D-printing of Urethane-based Photoelastomers for Vascular Tissue Regeneration

The mechanical properties of materials designated for vascular tissue replacement are of crucial importance. The elastic modulus, the tensile strength as well as the suture tear resistance have to be adjusted. Our approach is to use photopolymers for artificial vascular grafts. Via the layer-by-layer photopolymerization of suitable resin formulations as performed in additive manufacturing (AM) very complex structures are realizable. Hence AM offer the possibility to create cellular structures within the artificial grafts that might favor the ingrowth of new tissue. Commercially available urethane acrylates (UA) were chosen as base monomers since urethane groups are known to have good cell-adhesion behavior and poly-UAs show adequate mechanical performance. The mechanical properties of the photoelastomers can be tailored by addition of reactive diluents (e.g. 2-hydroxyethyl acrylate, HEA) and thiols (e.g. 3,6 dioxa-1,8-octane-dithiol) as chain transfer agents to comply with the mechanical properties of natural blood vessels. To examine the suture tear resistance a new testing method has been developed. Finally, a formulation containing 30 wt% UA and 70 wt% HEA complies with the mechanical properties of natural blood vessels, shows good biocompatibility in in-vitro tests and was successfully 3D-printed with digital light processing AM.